Project Summary Metastasis, the dissemination of cancer cells from the primary tumor to secondary sites, is the leading cause of cancer-related mortality. Metastasis is a multistep process, culminating in formation of clinically detectable tumor foci at distant organs. However, only a subpopulation of cancer cells within the primary tumor is capable of completing the entire metastatic cascade, as they must be dissemination-competent and contain tumor-initiating or stemlike capabilities. This deadly population of cells express high levels of MenaINV, a pro-metastatic isoform of the actin-regulatory protein Mena, and are capable of using doorways for intravasation and dissemination called Tumor Microenvironment of Metastasis (TMEM) sites. Preliminary studies lead to the hypothesis that the NF-kB signaling pathway may be a critical regulator of both the expression of Mena and stemness in cancer cells, promoting intravasation through blood vessels, through interactions with macrophages and through crosstalk with the Notch1 signaling pathway. These are very complex and context dependent signals. Therefore, while imaging tumors in live mice, these studies will use biosensors to determine the downstream consequences of NF-kB activation on Mena expression and stemness in tumor cells which are actively intravasating through TMEM. We will use two models of breast cancer metastasis, where human or mouse breast cancer cells are injected into mice and allowed to grow tumors. The mice express fluorescently labelled macrophages and endothelial cells. We will implant an imaging window over the tumor allowing for imaging at single-cell resolution of the tumor cells in the native tumor microenvironment. Depending on the experiment, the tumor cells will express biosensors to monitor activation of NF-kB signaling, stemness, and Mena promoter activity. Aim 1 will determine if activation of NF-kB signaling in tumor cells following collision with macrophages causes a distinct cellular phenotype compared to activation of NF-kB signaling without collision with macrophages. Whether inhibition of notch signaling through macrophage-tumor cell contact affects activation of NF-kB signaling will also be tested. Aim 2 will monitor the timing and order that the NF-kB, stemness, and Mena biosensors are activated to determine which signals promote the formation of the deadly intravasation competent and stem-like cells. The mice will then be treated with or without NF-kB inhibitors to examine if NF-kB signaling controls the activation of stemness or Mena expression, and if NF-kB signaling is required for intravasation of tumor cells through TMEM. Deciphering the mechanisms that produce a population of tumor cells that are both stem and intravasation- competent is critical to further our understanding of metastasis. The research environment in the Condeelis Lab at The Albert Einstein College of Medicine offers outstanding opportunities for training, collaborations, scientific discussions, and career development. The proposed studies and training plan developed in this proposal will instruct on mouse models of metastasis, biosensor development, and large volume high resolution intravital imaging techniques, ensuring mastery of all the tools needed to establish an independent lab.